IL-7 is required for T cell differentiation and mature T cell homeostasis and promotes pro-B cell proliferation and survival. Tyrosine phosphorylation plays a central role in IL-7 signaling. We identified by two-dimensional electrophoresis followed by anti-phosphotyrosine immunoblotting and mass spectrometry sixteen tyrosine phosphorylated proteins from the IL-7-dependent cell line D1. IL-7 stimulation induced the phosphorylation of the proteins STI1, ATIC and hnRNPH, involved in pathways related to survival, proliferation and gene expression, respectively, and increased the phosphorylation of CrkL, a member of a family of adaptors including the highly homologous Crk isoforms CrkII and CrkI, important in multiple signaling pathways. We observed an increased phosphorylation of CrkL in murine pro-B cells and in murine and human T cells. In addition, IL-7 increased the association of CrkL with the transcription factor Stat5, essential for IL-7 pro-survival activity. The selective tyrosine kinase inhibitor Imatinib. counteracted the IL-7 pro-survival effect in D1 cells and decreased CrkL phosphorylation. These data suggested that CrkL could play a pro-survival role in IL-7-mediated signaling. We observed that pro-B cells also expressed, in addition to CrkL, the Crk isoforms CrkII and CrkI and therefore utilized pro-B cells conditionally deficient in all three to evaluate the role of these proteins. The observation that the IL-7 pro-survival effect was reduced in Crk/CrkL conditionally-deficient pro-B cells further pointed to a pro-survival role of these adaptors. To further evaluate the role of these proteins, gene expression studies were performed in Crk/CrkL conditionally-deficient pro-B cells. IL-7 decreased the transcription of the receptor LAIR1, which inhibits B cell proliferation, in a Crk/CrkL-dependent manner, suggesting that the Crk family of proteins may promote pro-B cell proliferation. Our data contribute to the understanding of IL-7 signaling and suggest the involvement of Crk family proteins in pathways promoting survival and proliferation.

IL-7是T细胞分化和成熟T细胞稳态所必需的，并能促进pro-B细胞的增殖和存活。酪氨酸磷酸化在IL-7信号传导中起着核心作用。我们通过二维电泳，然后通过抗磷酸酪氨酸免疫印迹和质谱法鉴定了来自IL-7依赖细胞系D1的16种酪氨酸磷酸化蛋白。IL-7刺激诱导蛋白STI1，ATIC和hnRNPH的磷酸化，分别参与与存活，增殖和基因表达有关的途径，并增加了CrkL的磷酸化，CrkL是衔接子家族的成员，包括高度同源的Crk同种型。 CrkII和CrkI，在多种信号通路中很重要。我们观察到在鼠pro-B细胞以及鼠和人T细胞中CrkL的磷酸化增加。此外，IL-7增强了CrkL与转录因子Stat5的关联，后者对IL-7的生存前活性至关重要。选择性酪氨酸激酶抑制剂伊马替尼。抵消了D1细胞中IL-7的促存活作用，并降低了CrkL磷酸化。这些数据表明CrkL可以在IL-7介导的信号传导中发挥促生存作用。我们观察到，除了CrkL外，pro-B细胞还表达了Crk异构体CrkII和CrkI，因此利用了在所有三个条件上都存在缺陷的pro-B细胞来评估这些蛋白质的作用。在Crk / CrkL有条件缺陷的pro-B细胞中IL-7的促生存作用降低的观察结果进一步表明，这些衔接子具有促生存作用。为了进一步评估这些蛋白质的作用，基因表达研究是在Crk / CrkL条件缺陷的pro-B细胞中进行的。IL-7以Crk / CrkL依赖的方式降低了受体LAIR1的转录，从而抑制了B细胞的增殖，这表明Crk家族的蛋白质可能促进pro-B细胞的增殖。我们的数据有助于了解IL-7信号，并提示Crk家族蛋白参与促进存活和增殖的途径。